Method for the preparation of polymeric micelle via phase separation of block copolymer

ABSTRACT

A method for preparing a biodegradable polymeric micellar composition using liquid polyethylene glycol as a phase separation medium. The present invention also provides an efficient method to effectively incorporate a hydrophobic drug into a polymeric micelle in a polyethylene glycol separating medium.

This application is based on PCT /KR01/00765, which claims priority,based on a Korean patent application No. 2000-25256 filed on May 12,2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides a method for preparation polymericmicelles and use thereof in pharmaceutical applications. The micelle,which is used as a carrier for hydrophobic drugs, is prepared via phaseseparation of a biodegradable polymeric composition containing a blockcopolymer having a hydrophilic poly(alkylene glycol) component and ahydrophobic biodegradable polymer component suspended in a poly(ethyleneglycol) medium.

2. Related Art

Many important drugs are hydrophobic and have limited solubility inwater. In order to attain the expected therapeutic effect from suchdrugs, it is usually required that a solubilized form of the drug beadministered to a patient. Therefore, solubilization of a poorly watersoluble drug is key technology in the preparation of a formulation fororal or parenteral, especially intravenous, administration of the drug.Common methods used for solubilization of poorly water soluble drugsare: i) dissolving the drug in a co-solvent of a water-miscible organicsolvent and water; ii) modifying the drug to its salt form which issoluble in water; iii) forming a soluble drug-complex using a complexingagent; and iv) micellizing the drug in an aqueous medium with asurfactant. Leon Lachman, “The Theory and Practice of IndustrialPharmacy”, Lea & Febiger, Philadelphia, 1986.

Solubilization methods using surfactants, not requiring any changes inthe chemical structure of a drug, have been widely used to solubilizevarious drugs. Non-ionic surfactants, e.g. polyoxyethylene sorbitanfatty acid esters(Tween®) and polyoxyethylene alkyl ethers(Brij™ orMyrj™), are commonly used as the surface active agents. European PatentEP 0645145 discloses a method of solubilizing a typical poorly watersoluble drug, paclitaxel, by use of Cremophor EL™, a polyoxyethylenecastor oil derivative. However, the use of these surfactants, isrestricted due to toxic side effects such as hypersensitivity. They havelimitations in that their poor ability to stabilize micelles can causeprecipitation of the drug when the micellar solution is either stored oris to remain in place for an extended period of time.

Polymeric micelles have been recently investigated as potential carriersfor hydrophobic drugs. Eur. J. Pharm. Biopharm. 48(1999) 101-111.Polymeric micelles are characterized by a core-shell structureconsisting of hydrophobic inner core and hydrophilic outer shell. Apoorly water soluble drug is entrapped within the hydrophobic core ofthe micelle. There are two typical methods of entrapping a poorly watersoluble drug in the hydrophobic core of a micelle: a) a block copolymerand a poorly water soluble drug are dissolved in a water-miscibleorganic solvent, such as ethanol or N,N-dimethyl formamide(DMF), and thesolution is dialyzed in water (Dialysis Method); and b) a drug solutionof a water-immiscible organic solvent, such as dichloromethane orchloroform, is added to an aqueous polymeric solution and the organicsolvent is evaporated from the solution mixture (O/W Emulsion-SolventEvaporation Method).

Yokoyama et al. discloses methods of incorporating a poorly watersoluble drug into the inner core of a polymeric micelle using an A-Btype diblock copolymer composed of a hydrophilic methoxypolyethyleneglycol block(A) and a hydrophobic polyamino acid(B). See U.S. Pat. Nos.5,510,103 and 5,449,513. According to those patents, an aqueous micellarsolution of the diblock copolymer and an organic solvent solution of thehydrophobic component are prepared in separate containers. The twosolutions are then mixed and simply stirred, heated or sonicated toincorporate the hydrophobic drug into the polymeric micelles. Theaqueous polymer solution and the drug solution in DMF are mixed togetherand the mixture is dialyzed against an excess of water. These methodsrequire preparing an aqueous micellar solution prior to incorporating adrug into the polymeric micelle. See also, G. Kwon, et al., Blockcopolymer micelles for drug delivery: loading and release ofdoxorubicin, J. Contr. Rel. 48(1997) 195˜201; G. Kwon, et al., Physicalentrapment of Adriamycin in AB block copolymer micelles, Pharm. Res.12(1995) 192˜195.

X. Zhang et al. reported that a polymeric micelle prepared with adiblock copolymer of poly(lactic acid) and monomethoxy poly(ethyleneglycol) was useful as a carrier of paclitaxel. X. Zhang et al., Int. J.Pharm. 132(1996) 195-206. Shin et al. disclose a solubilization methodfor indomethacin using a diblock copolymer of poly(ethylene glycol) andpolycaprolactone. I. Gyun Shin et al., J. Contr. Rel., 51(1998) 13-22.In these methods, a poorly water soluble drug is incorporated in apolymeric micelle wherein the polymers are biocompatible andbiodegradable. According to their methods, a drug and a block copolymerare dissolved together in an organic solvent, especially in awater-miscible organic solvent, such as tetrahydrofuran or dimethylformamide. The polymeric micelles are prepared by first dialyzing thesolution in water and then freeze-drying the aqueous micellar solution.Alternatively, a solution of a polymer and drug in a water-miscibleorganic solvent, acetonitrile, is prepared. The organic solvent isslowly evaporated to give a homogeneous drug-polymer matrix and thematrix is then dispersed in an aqueous medium at about 60° C. to formthe polymeric micelles. It is stated that a polymeric micelle containingthe drug cannot be formed if an organic solvent other than anacetonitrile, such as chloroform, dichloromethane, ethyl acetate,acetone, methanol, ethanol, or tetrahydrofuran is used for dissolvingthe drug and polymer. The aqueous polymeric micellar solutions areprepared by heating, ultrasonic treatment, vortexing or mechanicalmixing.

As described above, conventional solubilization methods for poorly watersoluble drugs using polymeric micelles employs complicated stepsincluding formation of an aqueous polymeric micellar solution containinga poorly water soluble drug, followed by preparation of a freeze-driedpowder. Moreover, the powdered product must then be reconstituted so itis not possible to store the product in an aqueous solution for aprolonged period of time because of the hydrolyzable and biodegradablecomponents in the polymer. Another disadvantage is that this method cannot be applied to a polymer having a melting temperature below about 50°C. Furthermore, all existing methods for incorporating a drug into themicelle require using an organic solvent and preparing the polymericmicelles in an aqueous medium. It is very difficult to completelyeliminate the organic solvent in the process of preparing a polymericmicelle or incorporating a drug into the micelle. In addition, theremaining organic solvent decreases the stability of the micelle inwater and makes it difficult to control the release rate of the drug.

SUMMARY OF THE INVENTION

The present invention discloses a preparation method for preparation ofa non-aqueous polymeric micellar system without the use of significantamounts of an organic solvent which may have toxic side effects andrequire removal by evaporation. The present invention provides a methodfor preparing a polymeric micellar composition wherein a hydrophobicdrug is effectively incorporated, via phase separation of abiodegradable polymeric composition containing a block copolymer havinga hydrophilic poly(alkylene glycol) component and a hydrophobicbiodegradable polymer component suspended in a poly(ethylene glycol)medium, into the micelle.

The block copolymer is mixed together with a hydrophobic drug in theliquid polyethylene glycol. A solution of the polymer and the drug isthen obtained by heating the mixture. The solution is then slowly cooledand polymeric micelles having a core-shell structure form in thesolution via phase separation of the block copolymer from the liquidpoly(ethylene glycol). The terms poly(ethylene glycol), polyethyleneglycol, or PEG, as used herein, are interchangeable and shall also bedeemed to include derivatives of PEG unless otherwise specificallystated. Such derivatives will be more specifically described in thedisclosure that follows. Since only the hydrophilic component block andnot the hydrophobic component block of the copolymer has an affinity orattraction for the poly(ethylene glycol) matrix, the block copolymerforms a core-shell structure wherein the hydrophobic biodegradablepolymer block occupies the inner core and the hydrophilic poly(alkyleneglycol) block forms the outer shell in the poly(ethylene glycol) mediumor carrier.

The present invention uses liquid polyethylene glycol as a medium formixing and solubilization of a hydrophobic drug and thehydrophilic/hydrophobic copolymer, followed by phase separation of thepolymeric micelle which provides for a one step process of preparing thepolymeric micelle containing a poorly water soluble drug. In contrast,conventional methods employ two steps: 1) a polymeric micelle is formedin an aqueous media and 2) a poorly water soluble drug is incorporatedinto the micelle in the aqueous polymer solution.

The present invention also provides a method of incorporating a poorlywater soluble drug into a polymeric micelle having a core-shellstructure using liquid polyethylene glycol as a phase separation medium,removing the liquid polyethylene glycol and freeze-drying the resultingmicellar solution.

If desired, a biocompatible water-miscible organic solvent may be addedto the composition of the present invention to facilitate bettersolubility of the drug. The amount of organic solvent added depends onthe solubility of the drug, and the preferred content of the solvent isless than 50 wt % based on the amount of poly(ethylene glycol) or itsderivatives.

The present invention further provides an efficient method toeffectively incorporate a hydrophobic drug into a polymeric micelle in apolyethylene glycol separating medium. Aqueous solutions of micellesfrom which the polyethylene glycol has been removed can be filtered tosterilize them, freeze-dried and stored as a stable powder formulation.Furthermore, the composition can easily be reconstituted as a solutionand injected into the body and is therefore is useful for theintravenous administration of poorly water soluble drugs.

Additional features and advantages of the invention will be apparentfrom the detailed description which follows, taken in conjunction withthe accompanying examples, which together illustrate, features of theinvention.

DETAILED DESCRIPTION

Reference will now be made to the exemplary embodiments and specificlanguage will be used herein to describe the same. It will neverthelessbe understood that no limitation of the scope of the invention isthereby intended. Alterations and further modifications of the inventivefeatures illustrated herein, and additional applications of theprinciples of the invention as illustrated herein, which would occur toone skilled in the relevant art and having possession of thisdisclosure, are to be considered within the scope of the invention.

The present invention is directed to a method for preparing a polymericmicelle having a core-shell type structure using liquid polyethyleneglycol as a phase separation medium and a method of incorporating apoorly water soluble drug into the polymeric micelle. A polymericmicelle composition, in a dry-state, is obtained by dialyzing thepolymeric micellar PEG solution, containing a drug or not, against waterto remove the PEG followed by freeze-drying the resulting solution.

The composition containing an amphiphilic block copolymer having ahydrophilic poly(alkylene glycol) component and a hydrophobicbiodegradable polymer component dispersed or suspended in apoly(ethylene glycol) medium are disclosed in copending PCT/KR00/00885,hereby fully incorporated by reference (which has been filed in the U.S.as Ser. No. 09/807,487). The amphiphilic block copolymer comprises ahydrophilic poly(alkylene glycol) component and a hydrophobicbiodegradable polymer component. The polyalkylene glycol suitable as thehydrophilic component in the block copolymer of the present invention isa member selected from the group consisting of polyethylene glycol,monoalkoxy polyethylene glycol, or monoacyloxy polyethylene glycol,wherein the molecular weight of the polyalkylene glycol is preferablywithin the range of 1,000˜20,000 Daltons.

The hydrophobic biodegradable polymer component of the copolymer of thepresent invention is a member selected from the group consisting ofpolylactides, polycaprolactone, copolymers of lactide and glycolide,copolymers of lactide and caprolactone, copolymers of lactide and1,4-dioxan-2-one, polyorthoesters, polyanhydrides, polyphosphazines,poly(amino acid)s and polycarbonates. Preferably, the hydrophobicbiodegradable polymer component of the copolymer of the presentinvention is a member selected from the group consisting of apolylactide, polycaprolactone, a copolymer of lactide and glycolide, acopolymer of lactide and caprolactone, and a copolymer of lactide and1,4-dioxan-2-one. The molecular weight of the hydrophobic biodegradablepolymer component is preferably within the range of 1,000˜20,000Daltons, and more preferably within the range of 1,000˜10,000 Daltons.

The amphiphilic block copolymer of the present invention may be an ABtype diblock or an ABA or BAB type triblock copolymer comprising ahydrophilic poly(alkylene glycol) A-block component (A) and ahydrophobic biodegradable polymer B-block component(B), which formmicelles in an aqueous medium, and are dissolved or mixed homogeneouslyin a poly(ethylene glycol) medium.

The amphiphilic block copolymers can be prepared according to methodsdescribed in U.S. Pat. Nos. 5,683,723 and 5,702,717, hereby fullyincorporated by reference. For example they may be prepared via ringopening bulk polymerization of one of the monomers, such as a lactide,caprolactone, 1,4-dioxan-2-one, or a glycolide, with a polyethyleneglycol derivative in the presence of stannous octoate as a catalyst.Block copolymers having a poly(amino acid) block are prepared byreaction of an amino acid N-carboxy anhydride with a polyethylene glycolderivative. The hydrophilic polyethylene glycol block is preferably inthe range of 30˜70% by weight of the block copolymer, and mostpreferably 40˜60% by weight.

The liquid polyethylene glycol used for the phase separation medium inpreparing polymeric micelles (containing a poorly water soluble drug) ofthe present invention is preferably selected from the group consistingof dihydroxy, monoalkoxy, monoacyloxy, dialkoxy, or diacyloxypolyethylene glycol having a molecular weight of 200˜20,000 Daltons anda melting temperature of less than 65° C. More preferably, the liquidpolyethylene glycol is selected from the group consisting of dihydroxypolyethylene glycol, dialkoxy polyethylene glycol, and diacyloxypolyethylene glycol which are liquid at a temperature of 0˜40° C. andhas a molecular weight of 200˜20,000 Daltons, preferably 200˜10,000Daltons, and most preferably 200˜1,000 Daltons. Water, or an aqueoussolution, can be added to the liquid polyethylene glycol to facilitatephase separation of the block copolymer micelles. Preferably the amountadded will be less than 10% by weight of the liquid polyethylene glycolsolution.

A small amount of an organic solvent can be added to facilitate thesolubility of a poorly water soluble drug in the liquid polyethyleneglycol that is used for the phase separation medium. The solvent shouldbe biocompatible and easily eliminated by evaporation or dialysis. Forexample, ethanol, acetic acid, or acetone can be used as the solvent.Ethanol or acetic acid is the preferred selection for this purpose. Theamount added is preferably 0.1˜20% and is most preferably less than 10%by weight of the amount of polyethylene glycol used for the phaseseparation medium. Such amounts of organic solvents are considered, bydefinition herein, to be insignificant amounts when compared to theamount polyethylene glycol liquid medium.

Any drug having a water solubility of less than 10 mg/ml can be used asthe “hydrophobic drug” or “poorly water soluble drug” to be incorporatedin the polymeric micelle of the present invention. Examples ofhydrophobic drugs that can be used include anticancer agents,antiinflammatory agents, antifungal agents, antiemetics,antihypertensive agents, sex hormones, and steroids. Typical examples ofthe hydrophobic drugs are: anticancer agents such as paclitaxel,camptothecin, doxorubicin, daunomycin, cisplatin, 5-fluorouracil,mitomycin, methotrexate, and etoposide; antiinflammatory agents such asindomethacin, ibuprofen, ketoprofen, flubiprofen, dichlofenac,piroxicam, tenoxicam, naproxen, aspirin, and acetaminophen; antifungalagents such as itraconazole, and ketoconazole; sex hormones such astestosterone, estrogen, progestone, and estradiol; steroids such asdexamethasone, prednisolone, and triamcinolone; antihypertensive agentssuch as captopril, ramipril, terazosin, minoxidil, and parazosin;antiemetics such as ondansetron and granisetron; antifungal agents suchas amphotericin, metronidazole, and fusidic acid; cyclosporine; andbiphenyl dimethyl dicarboxylic acid. The present invention isparticularly useful for administering anti-cancer drugs such aspaclitaxel, taxotane, doxorubicin, cisplatin, carboplatin, 5-FU,etoposide, and camptothecin; sex hormones such as testosterone,estrogen, and estradiol; steroids such as triamcinolone acetonide,hydrocortisone, dexamethasone, prednisolone, and betamethasone;cyclosporine; and prostagladins.

According to a preferred embodiment of the present invention, apolymeric micelle is prepared as follows:

1) Dissolving of the amphiphilic block copolymer: The amphiphilic blockcopolymer is added to liquid polyethylene glycol to form a mixture. Themixture is heated and/or stirred until a solution is obtained.

2) Cooling and/or stirring of said solution, thereby forming a polymericmicelle by phase separation from the liquid polyethylene glycol whichserves as a phase separation medium.

3) Dialyzing the polymeric micellar containing composition formed instep (2), in liquid polyethylene glycol against excess water to removethe liquid polyethylene glycol that was used as a phase separationmedium.

4) freeze-drying the dialyzed aqueous solution to give a polymericmicellar composition in a fine powder state.

According to the present invention, a polymeric micelle containing ahydrophobic drug is prepared by dissolving the drug together with theamphiphilic block copolymer in the liquid polyethylene glycol in step(1) as described above. At a temperature of 30˜100° C., the drug and theamphiphilic block copolymer can be easily dissolved in the liquidpolyethylene glycol. If a small amount of organic solvent, such asethanol or acetic acid, is used to facilitate the solubility of ahydrophobic drug, the solution of step (1) is further slowly stirred ata temperature of 30˜100° C. to evaporate the organic solvent before thesolution is cooled in step (2). In any case, a drug containing polymericmicelle composition in the state of a fine powder is obtained bydialyzing the polymeric micellar solution against excess water followedby freeze-drying the resulting solution according to steps (3) and (4).Before dialyzing the polymeric micellar containing composition, thepolymeric micellar containing solution can be diluted with distilledwater to facilitate dialysis.

For the pharmaceutical use of the polymeric micelle prepared by thepresent invention, the dialyzed composition containing a poorly watersoluble drug obtained in step (3), is filtered through a membrane filterhaving a pore size of 0.22˜0.80 μm to sterilize the composition and thenfreeze-dried in an aseptic environment in step (4). When the blockcopolymer is dissolved in the liquid polyethylene glycol, the blockcopolymer content of the combined copolymer/polyethylene glycolcomposition is preferably 1˜50% by weight, and more preferably 10˜40% byweight. The poorly water soluble drug content in the polymeric micelleis preferably 0.1˜20% by weight based on the total weight of the drugand the block copolymer, and most preferably 1˜15% by weight. Thestabilizer, such as mannitol, sorbitol, lactose, or sucrose, can beadded to increase the stability of the freeze-dried micelle of thepresent invention. A stabilizer can be added in an amount of 0.1˜200% byweight based on the total weight of the drug and the block copolymer.The polymeric micelle prepared according to the present invention has adiameter of 10˜500 nm, preferably 10˜20 nm, and the micellar compositionwhen dispersed in saline can be used as a carrier for poorly a watersoluble drug via various routes: injectable(iv, im, sc); oral; and nasalroutes.

While the following examples are provided for the purpose ofillustrating certain aspects of the present invention, they are not tobe construed as limiting the scope of the appended claims.

EXAMPLES Example 1 Preparation of a mPEG-PLA Diblock Copolymer

2 g of monomethoxy polyethylene glycol (mPEG with a molecular weight of2,000 Daltons) was added to a round-bottomed flask and dried at atemperature of 100° C. while under vacuum (0.2 torr). Into the flask wasadded 2 g of lactide and 0.02 g of stannous octoate(catalyst). Thismixture was stirred for 6 hours at 120° C. under nitrogen flow. Thereaction product was cooled to room temperature and dissolved in 10 mlof dichloromethane. The solution was then poured into cold anhydrousether (−10˜0° C.) to precipitate the polymers, namely, diblockcopolymers of monomethoxy polyethylene glycol and polylactide(mPEG-PLA).The precipitated polymers were dried at 30° C. under vacuum (0.1 mmHg)for 48 hours.

Example 2 Preparation of a mPEG-PLGA Diblock Copolymer (LA/GA=7/3)

A diblock copolymer of monomethoxy polyethylene glycol andpoly(lactide-glycolide) (mPEG-PLGA) was prepared by the same method asin Example 1, using 2 g of monomethoxy polyethylene glycol(molecularweight of 2,000 Daltons), 0.7 g of lactide, and 0.3 g of glycolide inthe presence of 0.01 g of stannous octoate as a catalyst.

Example 3 Preparation of a mPEG-PLDO Diblcok Copolymer (LA/DO=5/5)

A diblock copolymer of monomethoxy polyethylene glycol andpoly(lactide-p-dioxanone) (mPEG-PLDO) was prepared by the same method asin Example 1, using 2 g of monomethoxy polyethylene glycol(molecularweight of 2,000 Daltons), 0.5 g of lactide, and 0.5 g of1,4-dioxan-2-one in the presence of 0.01 g of stannous octoate as acatalyst.

Example 4 Preparation of a mPEG-PCL Diblock Copolymer

A diblock copolymer of monomethoxy polyethylene glycol andpolycaprolactone (mPEG-PCL) was prepared by the same method as inExample 1, using 2 g of monomethoxy polyethylene glycol (molecularweight of 2,000 Daltons), and 0.8 g of caprolactone in the presence of0.008 g of stannous octoate as a catalyst.

Example 5 Preparation of a mPEG-PLA Diblock Copolymer

A diblock copolymer of monomethoxy polyethylene glycol and polylactide(mPEG-PLA) was prepared by the same method as in Example 1, using 2 g ofmonomethoxy polyethylene glycol (molecular weight of 5,000 Daltons), and1.8 g of lactide in the presence of 0.018 g of stannous octoate as acatalyst.

Example 6 Preparation of a Polymeric Micelle of mPEG-PLA

(1) Formation of a Polymeric Micelle

A 1 g sample of the diblock copolymer prepared in Example 1 (molecularweight: mPEG-PLA=2,000-1,800 Daltons) was mixed with 4 g of liquidpolyethylene glycol (Mw: 600 Daltons) and stirred for 30 minutes at 80°C. to obtain a solution. The solution was then slowly cooled to roomtemperature (25° C.) for 1 hour to obtain a composition of polymericmicelles formed in the liquid polyethylene glycol.

(2) Separation of the Polymeric Micelle

The composition obtained from the above step (1) was diluted with 4 mlof distilled water and the aqueous solution was then put into a dialysisbag. The liquid polyethylene glycol was removed from the solution bydialyzing the solution against water for 12 hours. An aqueous polymericmicellar solution was obtained.

(3) Sterilization and Drying

The dialyzed aqueous micellar solution obtained from the above step (2)was then filtered through a membrane filter, having a pore size of 0.22μm, to sterilize it and it was then freeze-dried in an asepticenvironment.

Comparative Example 1 A Polymeric Micelle of mPEG-PLA

According to a method described in U.S. Pat. No. 5,510,103, a polymericmicellar solution was prepared by dissolving 1 g of the diblockcopolymer prepared in Example 1(molecular weight: mPEG-PLA=2,000-1,800Daltons) in enough distilled water to give a concentration of 0.05%(w/v). The polymeric micellar composition in powder form was obtained byfreeze-drying the aqueous micellar solution.

The polymeric micelles prepared in Example 6 and Comparative Example 1was dispersed in suitable amount of distilled water to give aconcentration of 0.1% (w/v) and the particle size of each micellarsolution was determined by a dynamic light scattering (DLS) method. Theaverage micelle size of each preparation was nearly the same: 40 nm forExample 6, and 35 nm for Comparative Example 1

Example 7 Preparation of a Polymeric Micelle of mPEG-PLA ContainingPaclitaxel

(1) Formation of the Polymeric Micelle

A mixture was formed by adding 0.9 g of the diblock copolymer preparedin Example 1 (molecular weight: mPEG-PLA=2,000-1,800 Daltons) and 0.1 gof paclitaxel to 2 g of liquid polyethylene glycol (Mw: 600 Daltons).The mixture was stirred at 60˜90° C. for 20 minutes to give a clearsolution. The solution was slowly cooled to room temperature (about 25°C.) for 1 hour. A polymeric micellar composition containing paclitaxelwas obtained by phase separation from the liquid polyethylene glycol.

(2) Separation of the Polymeric Micelle

The composition obtained from the step (1) above was diluted with 2 mlof distilled water and then put into a dialysis bag. The liquidpolyethylene glycol was removed from the solution by dialyzing againstwater for 12 hours, and an aqueous polymeric micellar solutioncontaining paclitaxel was obtained in the dialysis bag.

(3) Sterilization and Drying

The dialyzed aqueous solution obtained from the step (2) above wasfiltered through a membrane filter, with a pore size of 0.22 μm, tosterilize it, and then freeze-dried in an aseptic environment. Theaverage micelle size was 45 nm, and paclitaxel in the micelle was 9.8%(loading efficiency=98%) by weight based on the total weight of the drugand the block copolymer.

Comparative Example 2 Polymeric Micelles of mPEG-PLA ContainingPaclitaxel

A polymeric micellar composition containing paclitaxel was prepared by amethod described in U.S. Pat. No. 5,510,103, hereby incorporated byreference.

Step 1: Formation of a Polymeric Micelle

A polymeric micellar solution was prepared by dissolving 0.9 g of thediblock copolymer prepared in Example 1 (molecular weight:mPEG-PLA=2,000-1,800 Daltons) in 900 ml of distilled water to give aconcentration of 0.1% (w/v).

Step 2: Incorporation of a Drug

A 0.1 g of paclitaxel dissolved in 1 ml acetone was added into thecomposition obtained from the Step 1 above. The mixture was stirred for2 hours at 80° C. and then cooled to room temperature ( about 25° C.).

Step 3: Sterilization and Drying

The aqueous solution obtained from Step 2 above was filtered through amembrane filter with a pore size of 0.22 μm, to sterilize it, and wasthen freeze-dried in an aseptic environment giving a powder state of thepolymeric micellar composition.

The polymeric micelles prepared in Example 7 and Comparative Example 2were dispersed in a suitable amount of distilled water to give aconcentration of 0.1% (w/v) and the particle size of each micellarsolution was determined by a dynamic light scattering (DLS) method. Theaverage micelle size of each preparation was nearly the same: 55 nm forExample 7, and 50 nm for Comparative Example 2.

The amount of drug incorporated in each composition prepared in Example7 and Comparative Example 2 was also determined by a HPLC assay. Theamount of paclitaxel for the composition of Example 7 was 9.8% (loadingefficiency=98%) by weight based on the total weight of the drug and theblock copolymer, and 8.7% (loading efficiency=87%) for the compositionof Comparative Example 2. The polymeric micellar composition of thepresent invention exhibited a higher loading efficiency than thatprepared according to U.S. Pat. No. 5,510,103.

Example 8 Polymeric Micelles of mPEG-PLDO Containing Cyclosporine A

(1) Formation of a Polymeric Micelle

A mixture was prepared by adding 0.95 g of the diblock copolymerprepared in Example 3 (molecular weight: mPEG-PLDO=2,000-1,940 Daltons)and 0.05 g of cyclosporine A to a solution mixture consisting of 3.2 gof liquid polyethylene glycol (mw: 600 Daltons) and 0.8 g of ethanol.The mixture was slowly heated to a temperature of 90° C., whilestirring, for 30 minutes to give a solution. The solution was slowlycooled to room temperature (about 25° C.) for 1 hour, and a polymericmicellar composition containing cyclosporine A was obtained.

(2) Separation of the Polymeric Micelle

The composition obtained from the step (1) above was diluted with 4 mlof distilled water and then put into a dialysis bag. The liquidpolyethylene glycol was removed from the solution by dialyzing againstwater for 12 hours, and an aqueous polymeric micellar solutioncontaining cyclosporine A was obtained.

(3) Sterilization and Drying

The dialyzed aqueous solution obtained from the step (2) above wasfiltered through a membrane filter, with a pore size of 0.22 μm, tosterilize it, and then freeze-dried in an aseptic environment. Theaverage micelle size was 50 nm, and cyclosporine A in the micelle was4.8% (loading efficiency=96%) by weight based on the total weight of thedrug and the block copolymer.

Example 9

Polymeric Micelles of mPEG-PLA Containing Paclitaxel

A polymeric micellar composition containing paclitaxel was prepared bythe method described in Example 7 using the following ingredients:

mPEG-PLA (mw: 2,000-1,800 Daltons): 0.85 g paclitaxel: 0.15 g diethoxypolyethylene glycol (mw: 600 Daltons): 5.00 g

Example 10 Polymeric Micelles of mPEG-PLA Containing Paclitaxel

A polymeric micellar composition containing paclitaxel was prepared bythe method described in Example 8 using the following ingredients:

mPEG-PLA (mw: 2,000-1,800 Daltons): 0.85 g paclitaxel: 0.15 g dimethoxypolyethylene glycol (mw: 600 Daltons): 4.00 g ethanol: 1.00 g

Example 11 Polymeric Micelles of mPEG-PLA Containing Paclitaxel

A polymeric micellar composition containing paclitaxel was prepared bythe method described in Example 7 using the following ingredients:

mPEG-PLA (mw: 2,000-1,800 Daltons): 0.98 g paclitaxel: 0.02 g dimethoxypolyethylene glycol (mw: 300 Daltons): 4.00 g

Example 12 Polymeric Micelles of mPEG-PLA Containing Paclitaxel

A polymeric micellar composition containing paclitaxel was prepared bythe method described in Example 7 using the following ingredients:

mPEG-PLA (mw: 2,000-1,800 Daltons): 0.95 g paclitaxel: 0.05 gdiacetyloxy polyethylene glycol (mw: 300 Daltons): 4.00 g

Example 13 Polymeric Micelles of mPEG-PLA Containing Paclitaxel

A polymeric micellar composition containing paclitaxel was prepared bythe method described in Example 7 using the following ingredients:

mPEG-PLA (mw: 2,000-1,800 Daltons): 0.80 g paclitaxel: 0.10 gpolyethylene glycol (mw: 200 Daltons): 5.00 g

Example 14 Polymeric Micelles of mPEG-PLA Containing Cyclosporine A

A polymeric micellar composition containing cyclosporine A was preparedby the method described in Example 8 using the following ingredients:

mPEG-PLA (mw: 2,000-1,800 Daltons): 0.90 g cyclosporine A: 0.10 gdimethoxy polyethylene glycol (mw: 200 Daltons): 3.60 g acetic acid:0.40 g

Example 15 Polymeric Micelles of mPEG-PLDO Containing Testosterone

A polymeric micellar composition containing testosterone was prepared bythe method described in Example 7 using the following ingredients:

mPEG-PLDO (mw: 2,000-1,800 Daltons): 0.95 g testosterone: 0.05 gpolyethylene glycol (mw: 600 Daltons): 2.00 g

Example 16 Polymeric Micelles of mPEG-PLDO Containing Doxorubicin

A polymeric micellar composition containing doxorubicin was prepared bythe method described in Example 7 using the following ingredients:

mPEG-PLDO (mw: 2,000-1,800 Daltons): 0.90 g doxorubicin: 0.10 gpolyethylene glycol (mw: 600 Daltons): 2.00 g

Example 17 Polymeric Micelles of mPEG-PCL Containing a Prostaglandin

A polymeric micellar composition containing a prostaglandin was preparedby the method described in Example 8 using the following ingredients:

mPEG-PCL (mw: 2,000-1,800 Daltons): 0.95 g prostaglandin: 0.05 gpolyethylene glycol (mw: 600 Daltons): 3.50 g ethanol: 0.50 g

The particle size and drug loading efficiency of the polymeric micellesobtained in Examples 7 to 17 and Comparative Example 2 are set forth inthe following Table 1.

TABLE 1 Particle Content^(a)) Loading size^(c)) Drug (wt %)efficiency^(b)) (nm) Example 7 Paclitaxel 9.8 98 45 ComparativePaclitaxel 8.7 87 50 Example 2 Example 8 Cyclosporine A 4.8 96 50Example 9 Paclitaxel 14.1 94 50 Example 10 Paclitaxel 14.3 95 40 Example11 Paclitaxel 1.98 99 45 Example 12 Paclitaxel 4.9 98 45 Example 13Paclitaxel 18.8 94 50 Example 14 Cyclosporine A 9.6 96 45 Example 15Testosterone 4.8 96 45 Example 16 Doxorubicin 9.7 97 40 Example 17Prostaglandin 4.7 94 45 ^(a))Content = Drug (g)/[Polymer (g) + Drug (g)]× 100 ^(b))Loading efficiency = Loading amount (g)/Initial amount (g) ×100 ^(c))Particle size: Size of polymeric micelle containing drug

It is to be understood that the above examples are illustrative ofapplication of the principles of the present invention. Numerousmodifications and alternative arrangements can be devised withoutdeparting from the spirit and scope of the present invention. Thepresent invention has been described above in connection with theexemplary embodiments(s) of the invention. It will be apparent to thoseof ordinary skill in the art that numerous modifications can be madewithout departing from the principles and concepts of the invention asset forth in the claims.

1. A method for preparing a biodegradable polymeric micellar compositioncomprising the steps of: 1) mixing 5 to 95 wt % of a compositionconsisting essentially of an amphiphilic block copolymer comprising ahydrophilic poly(alkylene glycol) A block and a hydrophobic polymer Bblock component and 5 to 95 wt % of a phase separation medium consistingessentially of liquid poly(ethylene glycol) and optionally from 0.1 to10 wt % water, wherein the hydrophobic biodegradable polymer B blockcomponent is a member selected from the group consisting ofpolylactides, polycaprolactone, copolymers of lactide and glycolide,copolymers of lactide and caprolactone, copolymers of lactide and1,4-dioxan-2-one, polyorthoesters, polyanhydrides, polyphosphazines,poly(amino acid)s and polycarbonates, and heating the resulting mixtureuntil said block copolymer is dissolved in said liquid poly(ethyleneglycol); 2) cooling said solution causing said block copolymer toseparate from said phase separation medium thereby causing polymericmicelles formation in said solution by phase separation; 3) dialyzingsaid solution against an excess of water or an aqueous solution toremove the phase separation medium; and 4) freeze-drying said dialyzedaqueous solution thereby forming a polymeric micellar composition in apowdered state.
 2. The method of claim 1, wherein said phase separationmedium is a member selected from the group consisting of dihydroxypolyethylene glycol, dialkoxy polyethylene glycol and diacyloxypolyethylene glycol having a molecular weight within the range of 200 to10,000 Daltons and a melting temperature of less than 65° C.
 3. Themethod of claim 1, wherein said phase separation medium furthercomprises 0.1 to 10 wt % water.
 4. A method for preparing abiodegradable polymeric micellar composition which contains ahydrophobic drug, comprising the steps of: 1) mixing a hydrophobic drugand a composition consisting essentially of an amphiphilic blockcopolymer having a hydrophilic poly(alkylene glycol) A block componentand hydrophobic biodegradable polymer B block component and a phaseseparation medium consisting essentially of liquid poly(ethylene glycol)and optionally 0.1 to 10% by weight water and optionally 0.1 to 20% byweight, based on the total weight of the drug and the block copolymer, astabilizing compound selected from the group consisting of mannitol,sorbitol, sucrose and lactose, wherein the hydrophobic biodegradablepolymer B block component is a member selected from the group consistingof polylactides, polycaprolactone, copolymers of lactide and glycolide,copolymers of lactide and caprolactone, copolymers of lactide and1,4-dioxan-2-one, polyorthoesters, polyanhydrides, polyphosphazines,poly(amino acid)s and polycarbonates, and then heating the resultingmixture until said block copolymer is dissolved in said liquidpoly(ethylene glycol), wherein said amphiphilic block copolymer in themixture is 1 to 50% by weight, and said hydrophobic drug is 0.1 to 20%by weight, based on the total weight of the drug and the blockcopolymer; 2) cooling said solution causing said block copolymer toseparate from said poly(ethylene glycol) as polymeric micelles by phaseseparation; 3) dialyzing said solution against an excess of water or anaqueous solution to remove the liquid polyethylene glycol; and 4)freeze-drying said dialyzed aqueous solution thereby forming a polymericmicellar composition in a powdered state.
 5. The method of claim 4,wherein said liquid polyethylene glycol is a member selected from thegroup consisting of dihydroxy polyethylene glycol, dialkoxy polyethyleneglycol and diacyloxy polyethylene glycol and has a molecular weight of200 to 10,000 Daltons and a melting temperature of less than 65° C. 6.The method of claim 4, wherein distilled water is added to said phaseseparation medium in an amount of 0.1 to 10% by weight to facilitate thephase separation.
 7. The method of claim 4, wherein said hydrophobicdrug is selected from the group consisting of paclitaxel, cyclosporine,prostaglandin, doxorubicin, testosterone, cisplatin and camptothecin. 8.The method of claim 4, wherein a stabilizing compound selected from thegroup consisting of mannitol, sorbitol, sucrose, and lactose is added tothe mixture of step 1, in an amount of 0.1 to 20% by weight based on thetotal weight of the drug and the block copolymer.